Gene therapy for progressive muscular dystrophy using a new generation adenovirus vector and transposase

Duchenne肌营养不良症(DMD)是一种由肌营养不良蛋白基因缺陷引起的致命的进行性肌肉萎缩疾病。除了辅助依赖型腺病毒载体(HDAdV)外，没有其他病毒载体能够包装14kb的全长dystrophin基因，而且HDAdV由于其基因组中的大片段缺失而比老一代腺病毒载体更安全。我们已经获得了携带myc标记的小鼠dystrophin全长cDNA的HDAdV(HDAdV-myc-mFldys)。我们将其注射到7日龄的utroin/dystrophin双基因敲除小鼠(dKO小鼠)的多个近端肌肉中，这些小鼠通常表现出与人类DMD非常相似的症状，因为近端肌肉是DMD患者的器官。注射8周后，转导的dystrophin广泛表达，我们发现中央有核的肌纤维显著减少，dystrophin相关蛋白p-DG和a-肌聚糖(a-SG)以及神经元型一氧化氮合酶(NNOS)恢复。(NNOS)。注射dKO的小鼠体重增加，运动能力改善，寿命延长。使用HDAdV，我们可以治疗DMD模型小鼠，即使治疗基因被转移到多个骨骼肌中。我们的结果表明，多次肌肉注射携带全长dystrophin的HDAdV可以减轻DMD患者的症状和补偿丧失的功能。为了目标基因产物的长时间表达，我们尝试利用睡美人转座子系统和HDAdV将dystrophin基因整合到染色体中。然而，它揭示了HDAdV的DNA结构是线性的，它不适合睡美人-转座子系统的环状DNA。因此，将Dystrophin基因整合到染色体上还需要进一步的研究。

Duchenne muscular dystrophy(DMD) is a fatal progressive muscle wasting disease caused by defects in the dystrophin gene. No viral vector except the helper-dependent adenovirus vector(HDAdv) can package 14kb full-length dystrophin cDNA and HDAdv is considerably safer than old-generation adenovirus vectors due to the large-size deletion in its genome. We have generated HDAdv that carries myc-tagged murine full-length dystrophin cDNA(HDAdv-myc-mFLdys). We injected it into the multiple proximal muscles of 7-day-old utrophin/dystrophin double knockout mice (dko mice), which typically show symptoms quite similar to human DMD because the proximal muscles are organs affected in DMD patients. Eight weeks after injections, the transduced dystrophin was widely expressed and we found a significant reduction of centrally nucleated myofibers and the restoration of dystrophin associated proteins, p-dystroglycan (p-DG) and a-sarcoglycan (a-SG), as well as neuronal nitric oxide synthase. (nNOS). The injected dko mice also showed an increase in body weight, an improvement in motor performances, and prolonged lifespan. Using HDAdv, we could treat DMD model mice, even when the therapeutic gene was transferred into multiple skeletal muscles. Our results suggest that multiple intramuscular administrations of HDAdv carrying full-length dystrophin may reduce symptoms and compensate for lost functions in DMD patients.For the long expression of target gene product, we tried to integrate the dystrophin gene into chromosome by using the sleeping beauty-transposone system and HDAdv. However, it revealed that the DNA construct of HDAdv is linear and it does not fit the circular DNA of the sleeping beauty-transposone system. So, further study is necessary to integrate the dystrophin gene into chromosome.